CN114806904A

CN114806904A - Functional microorganism and preparation method and application thereof

Info

Publication number: CN114806904A
Application number: CN202110804640.8A
Authority: CN
Inventors: 吴鑫颖; 邱树毅; 肖甜甜; 胡鹏刚; 冯子娟; 吴君海; 黄永光; 王雪郦; 雷青青; 黄福星
Original assignee: Guizhou University
Current assignee: Guizhou University
Priority date: 2021-07-16
Filing date: 2021-07-16
Publication date: 2022-07-29
Anticipated expiration: 2041-07-16
Also published as: CN114806904B

Abstract

The invention discloses a functional microorganism and a preparation method and application thereof, wherein the functional microorganism comprises Kluyveromyces marxianus YK2021-2 and Lactobacillus casei LB20201 which are screened from Hmong's sour soup; the kluyveromyces marxianus YK2021-2 and the lactobacillus casei LB20201 are preserved, the kluyveromyces marxianus YK2021-2 is preserved in Guangdong province microbial strain preservation center at 7-5 months in 2021, and the preservation number is GDMCC NO: 61737, respectively; the lactobacillus casei LB20201 has been preserved in Guangdong province microorganism culture collection center in 2020, 6 and 14 days, with the preservation number GDMCC No. 61034; mixing the buckwheat sour soup with the fermented buckwheat sour soup to improve the flavor of the buckwheat sour soup; can effectively exert or strengthen the health-care functional components in the buckwheat raw material.

Description

Functional microorganism and preparation method and application thereof

Technical Field

The invention relates to the technical field of microbial fermentation, in particular to a functional microorganism and a preparation method and application thereof.

Background

In recent years, with the improvement of living standard and health consciousness of people, cereals and whole-cereal products are concerned by consumers in China, wherein buckwheat is a food crop which is internationally recognized as both medicine and food, and has been listed as a high-quality characteristic food crop in Guizhou, compared with other cereals such as glutinous rice, wheat, corn and the like, the buckwheat contains rich nutrient elements such as resistant starch, protein, fat, dietary fiber and the like, and functional components such as polyphenols, fagopol, active peptides and the like. At present, buckwheat foods are gradually becoming a novel health food which is popular with the public in the market, the buckwheat foods have the characteristics of unique flavor and high nutritional and health-care values (Gusko, 2021), and a large number of researches show that the fermentation can improve the nutritional value of buckwheat (Huangjun, 2019; Xiao Yong, 2019; Song et al.2020), such as antioxidant activity. The biotransformation caused by microbial fermentation is improved, so that a certain probiotic function is given to the traditional cereal food, which is a processing method of high value-added performance food. The polyphenols of buckwheat called as 'seventh nutrient' mainly comprise flavonoids, tannins, phenolic acids, anthocyanins and the like, have the effects of resisting oxidation, resisting cancer, reducing blood sugar, reducing blood fat and the like, and are compounds with potential health promotion effects.

From the development trend of grain deep processing in the global market at present, various fermented products prepared by taking grains as main raw materials through lactobacillus fermentation are an important way for expanding the consumption channels of grain food, such as lactobacillus casei LB20201 and application thereof (CN111676167A), the fermented products are mainly prepared by single strain fermentation, and the effect of the fermented products of mixed fungi is difficult to achieve.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a functional microorganism and a preparation method and application thereof, so that the buckwheat sour soup which is sour and refreshing in taste, rich in flavor and remarkably improved in contents of organic acid and volatile flavor compounds is obtained while health-care functional components in buckwheat raw materials are effectively exerted and enhanced.

The purpose of the invention is realized by the following technical scheme: a functional microorganism comprises Kluyveromyces marxianus YK2021-2, Lactobacillus casei LB 20201; the Kluyveromyces marxianus YK2021-2 and the lactobacillus casei LB20201 are preserved, the Kluyveromyces marxianus YK2021-2(Kluyveromyces marxianus) is preserved in Guangdong province microbial strain preservation center at 7-5 months in 2021, and the preservation number is GDMCC NO: 61737; the Lactobacillus casei LB20201(Lactobacillus casei) is preserved in Guangdong province microorganism culture collection center at 6-14 th of 2020, with the preservation number GDMCC NO: 61034 and the preservation period of 30 years.

Further, the volume ratio of the Kluyveromyces marxianus YK2021-2 to the Lactobacillus casei LB20201 is 1: 5.

Further, the screening method of the Kluyveromyces marxianus YK2021-2 comprises the following steps:

performing high-throughput sequencing on the traditional fermented white sour soup to determine dominant microorganisms;

then, according to a sequencing result, an YPD culture medium is prepared to separate and screen the dominant bacterial strain to obtain the screened and separated dominant bacterial strain;

then purifying the screened dominant strains, and culturing on a YPD culture medium to obtain single strain colonies of each purified dominant strain;

then extracting a sample from a single bacterial colony for sequencing, carrying out homology comparison, and simultaneously constructing a phylogenetic tree of the bacterial strain;

then respectively inoculating the single-bacterium suspension into the sticky rice soup for fermentation on the premise of determining the strain and phylogenetic tree to obtain a white sour soup fermentation liquor;

and (3) measuring the total acid and the pH value of the obtained white acid soup fermentation liquor, and analyzing the fermentation performance of the strain by combining sensory evaluation to screen out Kluyveromyces marxianus YK2021-2 with better fermentation performance in the traditional fermented white acid soup.

Furthermore, the screening method of the lactobacillus casei LB20201 adopts a screening method of the lactobacillus casei LB20201 and application thereof (CN 111676167A).

Further, a method for preparing a functional microorganism, comprising the steps of:

activation of Kluyveromyces marxianus YK 2021-2: selecting Kluyveromyces marxianus YK2021-2 frozen at-80 deg.C, inoculating into YPD agar culture medium with volume of Kluyveromyces marxianus YK2021-2 of 100 μ L, and activating at 30 deg.C for 48 hr; then, selecting an activated single colony, inoculating the single colony in a YPD liquid culture medium, and then, shaking the flask at the temperature of 30 ℃ to activate the single colony for 18 hours for later use;

activation of lactobacillus casei LB 20201: freezing and storing in-80 deg.C Lactobacillus casei LB20201, inoculating in MRS agar culture medium, wherein the volume of Lactobacillus casei LB20201 is 100 μ L, and activating at 37 deg.C for 12 hr; then picking the activated single colony to inoculate in MRS broth culture medium, and then shaking the flask to activate for 12h at 37 ℃;

the Kluyveromyces marxianus YK2021-2 after two times of activation and the lactobacillus casei LB20201 are respectively mixed according to the volume ratio of 1:5 to form the functional microorganism.

Furthermore, the initial bacterial liquid concentration of the Kluyveromyces marxianus YK2021-2 and the Lactobacillus casei LB20201 is 1 × 10 ⁸ CFU/mL。

Further, the functional microorganism is used for improving the functional components of the buckwheat sour soup in the fermentation process.

Further, the preparation method of the buckwheat sour soup is characterized by comprising the following steps: the method comprises the following steps:

s1: sample preparation

S11, preparing buckwheat flour: weighing dried sweet buckwheat seeds, crushing, and sieving with a 80-mesh sieve for later use;

s12, preparation of a buckwheat culture medium: weighing the buckwheat flour obtained in the step S11, dissolving in water to prepare a solution, wherein the buckwheat flour has a mass value of 3% of the volume value of the water, adding 70U/g alpha-amylase, and liquefying at 70 ℃ for 10 min; adding 560U/g saccharifying enzyme, and keeping temperature at 60 deg.C for 120 min; then 75mL of the heat-preserved solution is filled in a container to be used as a sample, and high-pressure steam sterilization is carried out for 15min under the conditions of 0.1Mpa and 115 ℃; then taking out and placing to room temperature;

s2: fermentation of

Inoculating functional microorganism in the buckwheat flour liquid culture medium obtained in the step S12, wherein the volume of the functional microorganism is 4% of the volume of the buckwheat flour liquid culture medium, and fermenting at the constant temperature of 33 ℃ for 74 h.

The invention has the beneficial effects that:

(1) the functional microorganisms are applied to the buckwheat enzymolysis liquid for fermentation, and the result shows that the functional microorganisms ferment buckwheat, so that the buckwheat sour soup is uniform and glossy in color, sour and refreshing and palatable, and rich in buckwheat flavor.

(2) The buckwheat is fermented by the functional microorganisms, so that the content of total flavonoids and total phenols in the buckwheat sour soup is increased. Researches on DPPH free radical clearance rate, hydroxyl free radical clearance rate and ABTS + free radical clearance rate of the buckwheat sour soup in the fermentation process of the functional strains prove that the antioxidant activity of the buckwheat sour soup is increased by fermenting buckwheat by the functional microorganisms.

(3) Fermenting semen Fagopyri Esculenti with functional microorganism, and detecting six organic acids including malic acid, lactic acid, succinic acid, tartaric acid, oxalic acid, and acetic acid in semen Fagopyri Esculenti sour soup

(4) Buckwheat is fermented by functional microorganisms, differential metabolites of 41 polyphenols are totally obtained before and after buckwheat is fermented, and the content increase of apigenin, 4-hydroxybenzaldehyde, African catechin and other substances is promoted by the mixed fermentation of the functional microorganisms; secondary metabolites of flavonoids and phenols such as morin, 3',4',5, 7-tetrahydroxyisoflavone, paeonin glucoside, delphinidin, catechin, vanillin, pelargonidin-3-O-glucoside and formononetin are very different in the buckwheat culture medium.

(5) Compared with the single-strain fermented buckwheat sour soup, the functional microorganism fermented buckwheat sour soup has the advantages that the antioxidant activity of buckwheat is increased, the content of functional components is increased, the content of organic acid and volatile flavor compounds is also obviously increased, and a certain theoretical basis is provided for the application of functional microorganisms and the development of buckwheat fermented foods.

Drawings

FIG. 1: a reducing sugar content change diagram in the buckwheat fermentation process;

FIG. 2 is a schematic diagram: a graph of total acid content change during buckwheat fermentation;

FIG. 3: a pH value change diagram in the buckwheat fermentation process;

FIG. 4: a graph of the change of the content of total flavonoids in the fermentation process of buckwheat;

FIG. 5: a graph of the change of the total phenol content in the fermentation process of buckwheat;

FIG. 6: an antioxidant capacity test chart in the buckwheat fermentation process;

FIG. 7: sensory scoring chart in buckwheat fermentation process;

FIG. 8: a graph of the change of the content of organic acid in the fermentation process of lactic acid bacteria;

FIG. 9: a graph of organic acid content change in the yeast fermentation process;

FIG. 10: a graph of organic acid content change in the mixed fermentation process;

FIG. 11: different types of volatile flavor substance content maps in the buckwheat fermentation process;

FIG. 12: a flavor substance PCA scoring scatter diagram (A) and a PLD-DA model scoring scatter diagram (B) in the lactic acid bacteria fermentation process; (ii) a

FIG. 13: a flavor substance PLS-DA model replacement test chart C in the fermentation process of the lactic acid bacteria;

FIG. 14: obtaining a dispersion point (E) by a dispersion point diagram (D) of the flavor substance PCA and a PLD-DA model in the yeast fermentation process;

FIG. 15: a flavor substance PLS-DA model replacement test chart F in the yeast fermentation process;

FIG. 16: obtaining a dispersion point (H) by a flavor substance PCA score scatter diagram (G) and a PLD-DA model in the mixed fermentation process;

FIG. 17: a flavor substance PLS-DA model replacement inspection chart I in the mixed fermentation process;

FIG. 18: thermography of flavor-substance differential metabolites during buckwheat fermentation;

FIG. 19: obtaining a scatter diagram by PCA (principal component analysis) of a sample before and after buckwheat fermentation;

FIG. 20: obtaining a scatter diagram by using an OPLS-DA model of a sample before and after buckwheat fermentation;

FIG. 21: an OPLS-DA displacement test result chart of the sample before and after buckwheat fermentation;

FIG. 22: QC sample extraction ion flow graph PART 1;

FIG. 23: QC sample extraction ion flow graph PART 2;

FIG. 24: QC sample extraction ion flow graph PART 3;

Detailed Description

The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, but the scope of the present invention is not limited to the following.

A functional microorganism comprises Kluyveromyces marxianus YK2021-2, Lactobacillus casei LB 20201; the kluyveromyces marxianus YK2021-2 and the lactobacillus casei LB20201 are preserved, the kluyveromyces marxianus YK2021-2(Kluyveromyces marxianus) is preserved in Guangdong province microbial strain preservation center at 2021 and 07 and 05 days, and the preservation number is GDMCCNO: 61737, respectively; the lactobacillus casei LB20201(Lactobacillus casei) is preserved in Guangdong province microorganism strain preservation center at 6-14 th of 2020, with the preservation number GDMCCNO: 61034; the Kluyveromyces marxianus YK2021-2 and the lactobacillus casei LB20201 Miao white sour soup are screened, and the volume ratio of the Kluyveromyces marxianus YK2021-2 to the lactobacillus casei LB20201 is 1: 5. Example 1:

a method for improving the function of buckwheat sour soup by functional microorganism fermentation comprises the following steps:

s1 sample preparation

s2 preparation of functional microorganism

S21, strain activation:

activation of Kluyveromyces marxianus YK 2021-2: selecting Kluyveromyces marxianus YK2021-2 frozen at-80 deg.C, inoculating into YPD agar culture medium, wherein the volume of Kluyveromyces marxianus YK2021-2 is 100 μ L, and activating at 30 deg.C for 48 hr; then, selecting an activated single colony, inoculating the single colony in a YPD liquid culture medium, and then, shaking the flask at the temperature of 30 ℃ to activate the single colony for 18 hours for later use;

the initial bacterial liquid concentrations of the Kluyveromyces marxianus YK2021-2 and the Lactobacillus casei LB20201 are both 1 × 10 ⁸ CFU/mL；

S22, strain mixing: mixing the Kluyveromyces marxianus YK2021-2 which is activated twice and the lactobacillus casei LB20201 according to the volume ratio of 1:5 to form a functional microorganism;

s3 fermented buckwheat sour soup

Then 4% (v: v) of the functional microorganism is inoculated in a buckwheat flour liquid culture medium, and then the buckwheat flour liquid culture medium is fermented for 74 hours at a constant temperature of 33 ℃.

Example 2

s1 sample preparation

s12, preparation of a buckwheat culture medium: weighing 3% (m: v) of the buckwheat flour obtained in the step S11, dissolving in water, adding 70U/g of alpha-amylase, and liquefying at 70 ℃ for 10 min; adding 560U/g saccharifying enzyme, and keeping temperature at 60 deg.C for 120 min; then 75mL of the solution is taken and filled in a triangular flask with 150mL to be used as a sample, and high-pressure steam sterilization is carried out for 15min under the conditions of 0.1Mpa and 115 ℃; then taking out and placing to room temperature;

s2, strain activation

Activation of Kluyveromyces marxianus YK 2021-2: selecting Kluyveromyces marxianus YK2021-2 frozen at-80 deg.C, inoculating into YPD agar culture medium, wherein the volume of Kluyveromyces marxianus YK2021-2 is 100 μ L, and activating at 30 deg.C for 48 hr; then, selecting an activated single colony, inoculating the single colony in a YPD liquid culture medium, and then, shaking the flask at the temperature of 30 ℃ to activate the single colony for 18 hours for later use; the Kluyveromyces marxianus YK2021-2 is 1 × 10 ⁸ CFU/mL；

S3 fermented buckwheat sour soup

The yeast YK2021-2 of Kluyveromyces marxianus after two times of activation is taken, then inoculated into a buckwheat flour liquid culture medium according to 4% (v: v), and then fermented for 74 hours at the constant temperature of 33 ℃.

Example 3:

s1 sample preparation

s2, strain activation

Activation of lactobacillus casei LB 20201: freezing and storing in-80 deg.C Lactobacillus casei LB20201, inoculating in MRS agar culture medium, wherein the volume of Lactobacillus casei LB20201 is 100 μ L, and activating at 37 deg.C for 12 hr; then picking the activated single colony to inoculate in MRS broth culture medium, and then shaking the flask to activate for 12h at 37 ℃; the initial bacterial liquid concentration of the lactobacillus casei LB20201 is 1 multiplied by 10 ⁸ CFU/mL；

S3 fermented buckwheat sour soup

The lactobacillus casei LB20201 which is activated twice is taken, and then inoculated in a buckwheat flour liquid culture medium according to 4 percent (v: v), and then fermented for 74 hours at the constant temperature of 33 ℃.

Chemical composition detection

The chemical components in the sample fermentation process in the embodiment are measured by adopting plant MRM (multi-target chemometrics) wide-target metabonomics technology, the change of main polyphenols in fermentation liquor is comprehensively and systematically analyzed before buckwheat fermentation and after inoculation of functional strain fermentation, and Pearson correlation analysis is carried out on the polyphenols differential metabolites and the antioxidant activity, so that the correlation between the antioxidant activity and the polyphenols is known.

(1) Content determination of reducing sugar, total acid, pH value, total flavone and total phenol

In order to know the difference of physicochemical indexes of buckwheat hydrolysate fermented by a single strain and buckwheat hydrolysate fermented by a mixed strain, the physicochemical indexes of total sugar, reducing sugar, total acid, total flavone, total phenol and the like in the buckwheat hydrolysate and the glutinous rice flour hydrolysate were measured, and the measurement results are shown in table 1.

(1.1) determination of total acids (in terms of lactic acid): refer to the national standard GB/T12456-2008 'determination of total acid in food' (Chinese food fermentation industry research institute, 2008); pH value: refer to the national standard GB5009.237-2016 (national food safety Standard food pH determination) (food fermentation industry research institute, 2016); reducing sugar: the results of reducing sugar measurement (Liu Cating et al, 2020) by the 3, 5-dinitrosalicylic acid method (DNS method) are shown in FIG. 1, the results of total acid measurement are shown in FIG. 1, and the results of pH measurement are shown in FIG. 3.

(1.2) determination of Total Flavonoids

The total flavone content is determined by the methods of national Yixiancui (article published by Yixiancui et al in 2019 in Food industry Science 40(12): 31-37) And national Wang Fan (article published by Wang F et al in 2017 in Food Science And Technology International 23(2): 119-127). Taking the concentration of the rutin solution as a horizontal coordinate and the light absorption value as a vertical coordinate to prepare a standard curve, wherein the regression curve equation is as follows: y ═ 0.0067x-0.0014 (R) ² 0.999). And (3) sample determination: accurately weighing 1.00mL of prepared sample to be measured by a pipette, adding purified water into a 10mL volumetric flask to fix the volume, measuring the absorbance according to the method, and calculating the content of corresponding total flavonoids according to a standard curve, wherein the measurement result is shown in FIG. 4.

(1.3) measurement of Total phenol content

The determination of the total phenol content refers to the method of XieYan and the like (the article published by XieYan and the like in 2009 in food research and development 30(03): 126-; the regression curve equation is: y0.0545 x-0.0005 (R) ² ＝0.999)。

And (3) determination of a sample: accurately measuring 0.50mL of prepared sample solution to be measured in a 50mL volumetric flask by using a pipette, fixing the volume by using purified water, measuring the absorbance according to the method, and calculating the content of corresponding total phenol according to a standard curve; the measurement results are shown in FIG. 5.

Table 1: physical and chemical indexes of buckwheat hydrolysate

As can be seen from Table 1, small amounts of total acids, total flavonoids and total phenols were detected in the buckwheat hydrolysate.

As can be seen from fig. 1: the mixed fermentation has the highest utilization rate of reducing sugar, and then the fermentation of Kluyveromyces marxianus YK2021-2, and the fermentation of Lactobacillus casei LB20201 has the lowest utilization rate of reducing sugar. In the buckwheat sour soup, when the mixed fermentation is carried out for 74 hours, the content of reducing sugar is 3.00mg/mL, and when the single strain of lactobacillus casei LB20201 is fermented for 74 hours, the content of reducing sugar is 10.29 mg/mL; in 12-24 h, the content of Kluyveromyces marxianus YK2021-2 and mixed fermentation reducing sugar in the buckwheat culture medium is obviously reduced, the strain has vigorous growth and metabolism activity in the process, the reducing sugar is used as a carbon source to provide nutrient substances for the growth of the strain, and the content of the reducing sugar is basically stable within 36-74 h.

As can be seen from fig. 2: the total acid content of mixed fermentation in the buckwheat fermentation process is obviously lower than that of single strain fermentation of lactobacillus casei LB 20201. But when the fermentation is carried out for 0-12 h, compared with the single-strain fermentation of lactobacillus casei LB20201, the total acid content is increased by mixed fermentation; and when the time is 36-74 hours, the total acid content in the mixed fermentation system is lower than that in the single strain fermentation of the lactobacillus casei LB 20201. The pH value change trend is similar to the total acid change trend, and the pH value of the buckwheat sour soup fermented by the single strain of lactobacillus casei LB20201 is the lowest; the saccharides in the buckwheat provide a large amount of nutrient substances for the growth of the lactobacillus casei LB20201, and promote the metabolism of the lactobacillus casei LB20201, so that the total acid content in the buckwheat sour soup is obviously increased.

As can be seen from fig. 4: the content of total flavone in the buckwheat liquid culture medium is 10.96 mu g/mL, and after single-strain fermentation and mixed-strain fermentation of functional microorganisms of Kluyveromyces marxianus YK2021-2 and Lactobacillus casei LB20201, the content of total flavone is 9.31 mu g/mL, 10.86 mu g/mL and 11.05 mu g/mL respectively. In the lactobacillus casei LB20201 fermentation system, the total flavone content fluctuates in the fermentation process and increases at 12h and 36h, which is that in the lactobacillus casei LB20201 fermentation process, macromolecular flavonoid substances are decomposed into small molecular flavonoid substances, and the small molecular flavonoid substances are further decomposed into flavonoid secondary metabolites by the enzyme action of the fermentation system, so that the total flavone content fluctuates in the fermentation process.

As can be seen from fig. 5: the total phenol content before buckwheat fermentation is 1.18 mug/mL, and after single strain fermentation and mixed strain fermentation of lactic acid and Kluyveromyces marxianus YK2021-2, the total phenol content is respectively as follows: 1.24. mu.g/mL, 1.21. mu.g/mL, 1.32. mu.g/mL. After buckwheat is fermented, the content of total phenols is increased, and total flavonoids are decomposed and metabolized into some phenolic substances under the action of microorganisms. Microbial metabolism in the grain can produce polyphenols, which increase the total phenol content during fermentation. Phenolic substances can be hydrolyzed and oxidized in fermentation liquor, so that the total phenol content of the functional microorganisms fluctuates in the fermentation process. And (5) fermenting for 74 hours, wherein the total phenol content in the mixed fermentation system is higher than that in the single strain fermentation. The study with Dingyufeng et al (refer to the article published by Dingyufeng et al in 2021 in food science 1-14) shows that during the fermentation process of grape ferment, microorganisms promote the release of phenols, and the total phenol content is basically consistent with the fluctuation change result.

(2) Measurement of antioxidant Capacity

(2.1) DPPH radical scavenging ability

1,1-diphenyl-2-picrylhydrazyl (1,1-diphenyl-2-picrylhydrazyl, DPPH) free radical scavenging ability determination reference to Liangxin et al (Liangxin et al, published in 2021 in food and fermentation industry 47(07): 175-;

hydroxyl radical scavenging ability

The hydroxyl radical scavenging ability was determined by the method of Liangxin et al (Liangxin et al, published in 2021 in food and fermentation industries 47(07): 175-;

(2.2)ABTS ⁺ free radical scavenging ability

ABTS + free radical scavenging capacity is determined by Dulong et al (Dulong et al, 2018, Food Chemistry 262(5): 21-29);

the results of the measurement of antioxidant ability are shown in FIG. 2.

As can be seen from fig. 6: the DPPH in the buckwheat liquid culture medium is 44.18%, after fermentation for 74 hours, the DPPH free radical clearance rate is obviously improved, the DPPH free radical clearance rates in Kluyveromyces marxianus YK2021-2, Lactobacillus casei LB20201 and a mixed fermentation sample are 83.57%, 85.27% and 85.40%, wherein the DPPH free radical clearance rates in the fermentation of Lactobacillus casei LB20201 and the mixed fermentation liquid are similar and higher than that in the fermentation of Kluyveromyces marxianus YK 2021-2. The protein in the buckwheat accounts for 8.51-18.87% (refer to the Master academic paper completed by Chenyajun in 2017 at the university of southern China), and the polyphenol and the polypeptide in the buckwheat are important material bases for the fermentation product to exert antioxidant activity, so that the Kluyveromyces marxianus YK2021-2 is used for improving the protein metabolism in the buckwheat during single-strain fermentation.

As can be seen from fig. 6: the clearance rate of hydroxyl free radical (-OH) is improved after buckwheat fermentation, the clearance rate of hydroxyl free radical in the buckwheat fermentation liquid culture medium is 71.11%, and after 74 hours of fermentation, 86.80% (Kluyveromyces marxianus YK2021-2 fermentation), 83.11% (Lactobacillus casei LB20201 fermentation) and 85.42% (mixed fermentation) are respectively carried out; the clearance rate of hydroxyl free radicals of Kluyveromyces marxianus YK2021-2 fermentation and mixed fermentation is obviously higher than that of Lactobacillus casei LB20201 fermentation.

As can be seen from fig. 6: when the fermentation is carried out for 74 hours, the ABTS + free radical clearance rates in Kluyveromyces marxianus YK2021-2, Lactobacillus casei LB20201 and mixed fermentation liquor are respectively 90.46%, 86.59% and 94.09%; ABTS in mixed fermentation broth ⁺ The clearance rate of free radicals is higher than that of Kluyveromyces marxianus YK2021-2 and Lactobacillus casei LB20201 fermentation.

To sum up: the buckwheat can be fermented by functional microorganisms, so that the antioxidant activity of the buckwheat is improved, wherein the increase of the antioxidant activity of mixed fermentation is better than that of single-strain fermentation.

(3) Determination of organic acid content in buckwheat fermentation process

A standard curve of the relationship between the peak area and the concentration of organic acids such as oxalic acid, tartaric acid, malic acid, lactic acid, acetic acid, succinic acid and the like is established by referring to the method of Wanghuling (Chinese brewing 39(06):196-203 by Wanghuling et al in 2020), the regression curve equation is detailed in Table 2, and the measurement results are shown in FIGS. 8-10.

Table 2:

serial number	Name(s)	Regression equation	Determining coefficients
					1	Oxalic acid	y＝22273x+55.067	0.9978
2	Tartaric acid	y＝3000.4x+14.49	0.9979
				3	Malic acid	y＝1404.9x+5.8238	0.9942
4	Lactic acid	y＝1093.3x+3.8524	0.9958
				5	Acetic acid	y＝1093x+7.8	0.9980
6	Succinic acid	y＝1059.3x+3.0381	0.9985

As can be seen from fig. 8: when the fermentation of the buckwheat starts, the acetic acid content is obviously increased, when the fermentation starts, the strain is vigorous, the lactobacillus casei LB20201 produces acetic acid mainly through glycolysis, and a small amount of acetic acid can also be produced in citrate circulation; in the buckwheat sour soup, lactobacillus casei LB20201 metabolizes succinic acid into tartaric acid mainly through citrate circulation, so that the content of tartaric acid in fermentation liquor increases along with the extension period of fermentation time when the fermentation liquor is fermented for 0-24 h, the contents of malic acid and succinic acid are reduced, and the contents of tartaric acid, malic acid and succinic acid are respectively 0.17mg/mL, 0.009mg/mL and 0.006mg/mL at 24 h. And in 24-60 h, the contents of malic acid, succinic acid and tartaric acid tend to be stable. After 60h, the content of tartaric acid is reduced, which is probably that lactobacillus casei LB20201 is in a decline stage in the later fermentation stage, the metabolic activity of the strain is reduced, succinic hydrolase is lacked, and tartaric acid is metabolized into other substances under the action of other enzymes. The content of lactic acid reaches 13.25mg/mL when the fermentation is carried out for 74 hours.

As can be seen from fig. 9: the lactic acid content of the Kluyveromyces marxianus YK2021-2 in the single strain fermentation process is reduced (0-60 h) along with the fermentation time, and is related to the combination of alcohol substances metabolized by the Kluyveromyces marxianus YK2021-2 in the fermentation process into esters. The tendency of acetic acid to change in buckwheat sour soup (fermentation of Kluyveromyces marxianus YK 2021-2): when the fermentation is started (0-24 h), the acetic acid content is increased along with the prolonging of the fermentation time, the acetic acid content tends to be stable after 24-36 h, the acetic acid content is reduced after 48h of fermentation, and the acetic acid content is increased after the end of the fermentation. In the fermentation process, the Kluyveromyces marxianus YK2021-2 metabolizes acetic acid, ethanol and the like in a large amount, the acetic acid and alcohol substances are combined into esters, and the content of the acetic acid metabolized by the Kluyveromyces marxianus YK2021-2 is close to the content of the acetic acid consumed in the fermentation liquor in 24-36 hours, so that the content of the acetic acid in the fermentation liquor tends to be stable in the time period, and the acetic acid is consumed in a large amount, so that the content of the acetic acid is reduced. After 48 hours, the acetic acid content is increased, the Kluyveromyces marxianus YK2021-2 is in the later fermentation stage, and a large amount of Kluyveromyces marxianus YK2021-2 cells are apoptotic, so that nutrients and enzymes in the cells are dissolved out in a large amount, the acetic acid content is increased, and the contents of tartaric acid, succinic acid, malic acid and the like are reduced.

As can be seen from fig. 10: the acetic acid change trend of the first 60h in the mixed fermentation process is basically consistent with the change trend of the lactobacillus casei LB20201 in the fermentation process, and the acetic acid content is similar to that of Kluyveromyces marxianus YK2021-2 fermentation after 60h, so that the content is increased. The lactic acid content is obviously lower than that of single-strain fermentation of lactobacillus casei LB20201, nutrition competition exists among mixed-strain fermentation strains, and components in the mixed-strain fermentation liquor are more complex to influence the metabolism of the strains or other compounds are generated through mutual reaction among metabolites. The tartaric acid content and the malic acid content in the mixed bacteria (12-60 h) are in opposite trend, and the succinic acid, tartaric acid and malic acid content is reduced after fermentation for 60-74 h, so that the content is reduced due to the generation of a large amount of flavor substances in the after-fermentation. The organic acid content in the mixed fermentation buckwheat is obviously improved.

(4) GC-MS determination of volatile flavor compounds

The functional microorganisms are applied to the buckwheat culture medium for fermentation, and influence on volatile flavor compounds in the buckwheat sour soup is achieved. The total detection of 8 types of volatile organic compounds in the buckwheat sour soup is 65 types, wherein the 65 types comprise 18 types of alcohols, 13 types of esters, 11 types of acids, 13 types of aldehydes, 5 types of ketones, 2 types of pyrazines, 1 type of furans and 2 types of other compounds, and the concentration of different types of volatile substances in a functional microorganism mixed strain and single strain fermentation system is shown in figure 7 along with the change of fermentation time; meanwhile, the major volatile flavor substances in the buckwheat fermentation process are analyzed by constructing a PLS model, and the specific analysis results are shown in figures 12-17.

Sample processing method

Head Space-Solid Phase Microextraction (HS-SPEM): putting 8.0mL of sample to be detected into a 20mL headspace bottle, adding 2.5g of sodium chloride and 5 μ L of internal standard (2-octanol, the concentration is 26.76 μ g/L), inserting an aged 50/30 μm CARR/PDMS/DVB extraction head into the headspace part of the sample bottle, adsorbing at 60 ℃ for 30min, taking out the adsorbed extraction head, inserting the extraction head into a gas chromatography injection port, desorbing at 250 ℃ for 3min, and starting an instrument to collect data.

And (3) GC-MS determination: a chromatographic column is an SH-Rtx-Wax capillary column (30m multiplied by 0.25mm multiplied by 0.25 mu m), carrier gas is high-purity helium gas (the flow rate is 2 mL. min < -1 >), an electron impact ionization (EI) ion source is adopted, the ion source temperature is 230 ℃, the electron energy is 70eV, and the scanning range is 35-350 amu.

Temperature programming conditions: initial temperature of chromatographic column at 40 deg.C for 3min, and temperature at 4 deg.C/min ^-1 Raising the temperature to 150 ℃ at a speed rate, keeping the temperature for 2min, and then raising the temperature to 8 ℃ for min ^-1 The temperature is increased to 230 ℃ at a speed, the temperature is kept for 6min, and the split-flow sample injection is not carried out.

Identification of volatile flavor compounds

The identification of aroma components is described by: (1) comparing with standard mass spectrum of NIST17s, NIST17-1, NIST17-2, SmartDatabase; (2) comparing the fragrance characteristics with those of the standard product; (3) the comparison with the Retention Index (RI) of a standard, and the comparison with the RIL reported in the literature, wherein the calculation method of RI is calculated by referring to the improved Kovats method (refer to Cates V E et al, published in 1963 in Journal of Chromatography 11(4): 472-.

Quantitative analysis of flavor compounds

Quantification by an internal standard method: and (3) obtaining the area of each chromatographic peak through an automatic integration program, calculating the peak area of the internal standard substance and the peak area ratio of each component in the sample, thereby quantifying the content of each flavor component, and measuring each group of samples for six times to obtain an average value.

Calculating the formula:

wherein Ci is the mass concentration (mu g/L) of the component i to be detected; WS is the concentration at which internal standard s is added (. mu.g/L); ai and As are respectively the peak areas of the component i to be measured and the internal standard compound s; v is the volume (L) of the sample to be measured; f is the relative mass correction factor of the component i to be measured to the internal standard s (the relative correction factor of each component i to be measured in the experiment is 1).

As can be seen from fig. 11: the detected 65 volatile compounds are consistent with the results of Jiangying (Jiangying completed in 2017 at Guizhou university is a Master's academic paper) research on buckwheat wine, And are significantly higher than the volatile flavor compounds in other buckwheat fermented foods (Zhangyun et al published in 2015 in food industry science 36(08): 222-; Wang A et al published in 2012 in Flavour And Fragrance Journal 27(1): 47-53). The concentration of total esters in the buckwheat sour soup is 342.93 mug/L. From fig. 12 to fig. 17, it can be seen that in the buckwheat sour soup, the contents of ester, alcohol, acid and aldehyde compounds in the functional strain mixed fermentation system are all significantly higher than those in single strain fermentation, and in the whole, these four compounds are the most abundant volatile organic compounds detected in the buckwheat sour soup; as can be seen from fig. 12 to 17: according to PCA, in the buckwheat sour soup, the single-strain fermentation of the functional microorganisms Kluyveromyces marxianus YK2021-2 and the lactobacillus casei LB20201 and the whole sample of mixed fermentation can be well distinguished among groups, which shows that the metabolites among the groups have better difference. The PLS model shows that in the buckwheat fermentation process, each sample is positioned in a 95% confidence interval, and the samples are distinguished very obviously. In buckwheat sour soup, functional microorganisms such as Lactobacillus casei LB20201, Kluyveromyces marxianus YK2021-2, and PLS model replacement test R of mixed fermentation group ² X、R ² Y、Q ² Respectively (0.952, 0.990, 0.977), (0.981, 0.990, 0.985), (0.983, 0.969, 0.960). The successful construction of the PLS model is demonstrated, the fitting degree and the prediction capability are high, and the method can be well explained in the fermentation process of functional microorganisms in the buckwheat fermentation mediumA metabolic difference of (a); based on the analysis results of the PLS model, metabolites with VIP value greater than 1 and p value less than 0.05 were used as differential metabolites in buckwheat fermentation process, and a total of 11 differential metabolites were identified, wherein 3 kinds of alcohols, 4 kinds of aldehydes, 2 kinds of acids and 2 kinds of esters, and the results are shown in FIG. 18. Compared with the buckwheat culture medium, the functional microorganism fermentation obviously increases the contents of ester, alcohol and aldehyde flavor substances in the buckwheat sour soup, and greatly improves the flavor of the buckwheat sour soup.

(5) Sensory evaluation

In order to examine the influence of the functional strains applied to the buckwheat culture medium for fermentation on the sensory quality of the buckwheat sour soup. The research consists of 15 sensory evaluators, the color, the internal form, the taste and the aroma of the buckwheat sour soup are evaluated respectively, the percentage system is adopted, the specific sensory evaluation standard is shown in a table 3, and the detailed sensory score in the buckwheat fermentation process is shown in a table 7.

TABLE 3 sensory evaluation index of buckwheat sour soup

As can be seen from FIG. 7, in the buckwheat culture medium, the sensory score of the single strain fermentation of the lactic acid bacteria is the lowest, when the fermentation is carried out for 0-60 h, the sensory score is increased along with the extension of the fermentation time, and when the fermentation is carried out for 60-74 h, the sensory score is reduced. In the fermentation process of the single lactobacillus strain, along with the prolonging of the fermentation time, the metabolism of the lactobacillus produces a large amount of organic acid, and the sour-sweet ratio in the buckwheat sour soup is disordered in the later period of fermentation, so that the sensory score is reduced. In the later stage of single-strain fermentation of the microzyme, the alcohol fermentation taste is heavier, and ester compounds are generated in the later stage of mixed-strain fermentation, so that the sensory score of the buckwheat sour soup is highest.

(6) Analysis of antioxidant component in buckwheat

Metabolite extraction

After the sample to be tested is vortexed for 30s, the sample is centrifuged for 15min at 4 ℃ and 12000 rpm; taking 1000 mu L of supernatant liquid to be placed in an EP tube, and drying by nitrogen blowing; then 500. mu.L of 50% methanol was added for reconstitution (containing internal standard); then vortex for 30s, and then ultrasonically process for 15min in an ice water bath; then centrifuging for 15min at 4 ℃ and 12000 rpm; filtering the supernatant with 0.22 microporous membrane, diluting the supernatant with 5 times of the extractive solution, swirling for 30s, taking into 2mL sampling bottle, and mixing 60 μ L of each sample to obtain QC sample; storing at-80 deg.C until detecting on machine.

UHPLC-MS/MS analysis

Chromatographically separating the objective compound by a Waters UPLC liquid chromatography column (ACQUITYUPLCHSST31.8 μm × 2.1 × 100mm) using EXIONLCSYSTEM (SCIEX) ultra performance liquid chromatography; the liquid chromatogram is characterized in that a phase A is a 0.1% formic acid aqueous solution, and a phase B is acetonitrile; the following analysis was performed with an elution gradient: 0-0.5 min, 98% A; 0.5-10 min, 98% -50% A; 10-13 min, 50% -5% A; 13.1-15 min, 5% -98% A. The flow rate was 0.4 mL/min ^-1 The column oven temperature was 40 ℃, the autosampler temperature was 4 ℃, and the sample injection volume was 2 μ L.

SciexQTrap6500 Mass Spectrometry instrument parameters: performing mass spectrometry in a Multiple Reaction Monitoring (MRM) mode using a SCIEX6500QTRAP + triple quadrupole mass spectrometer equipped with an Ion Drive Turbo VESI Ion source; the ion source parameters were as follows: the ion spray voltage is +5500/-4500V, the curtain gas is 35psi, the temperature is 400 ℃, the gas is 1:60psi, the gas is 2:60psi, and the DP is + -100V.

(7) Analysis of polyphenol components before and after buckwheat fermentation

As shown in fig. 22-24, the Extracted Ion Chromatograms (EICs) of the sample show that the analysis method adopted by the present invention makes all target compounds present symmetrical chromatographic peaks, and the chromatographic separation of each target compound is well realized.

As can be seen from FIG. 19, significant differentiation occurred between the samples before and after buckwheat fermentation, indicating that PCA can clearly distinguish the two groups before and after buckwheat fermentation, and that each group has good reliability. The Orthogonal Partial least squares Discriminant Analysis (OPLS-DA) of fig. 20 shows the differences between and within buckwheat sample groups, and as can be seen from the results of the OPLS-DA score plot, the two groups of samples are distinguished very significantly. The results of the replacement test of BCN group of buckwheat with the OPLS-DA model of BYM group are shown in FIG. 21,r in OPLS-DA model of sample before and after buckwheat fermentation ² X、 R ² Y、Q ² The values of (A) are 0.966, 0.999 and 0.897 respectively, and the values are close to 1, which shows that the model has good robustness, the model fitting is good, the over-fitting phenomenon does not exist, and the OPLS-DA model can well explain the difference between two groups of samples before and after buckwheat fermentation. With VIP ≥ 1.0 and p<0.05 as a standard, screening to obtain differential metabolites of 41 polyphenols in two groups of samples before and after buckwheat fermentation, wherein the differential metabolites of the main polyphenols before and after buckwheat fermentation are shown in table 4;

the content increase of apigenin, 4-hydroxybenzaldehyde, afzelechin and other substances is promoted by the mixed fermentation of functional microorganisms, the apigenin generates 4',5, 7-trihydroxyflavone mainly through cinnamoyl coenzyme A under the action of enzyme, and then is further metabolized into apigenin, and the afzelechin is generated mainly through leucine catabolism. The content of anthocyanin, quercetin, catechin, 3,4,5, 7-tetrahydroxy isoflavone is reduced in the fermentation process of the functional microorganism mixed bacteria, on one hand, the anthocyanin can be decomposed into the quercetin, the catechin, the epicatechin and the like in the fermentation process, and the substances of the quercetin, the catechin, the epicatechin and the like in the buckwheat sour soup can be further decomposed and metabolized, so that the content is reduced after the fermentation of the functional microorganism; on the other hand, in the fermentation process, the temperature in the fermentation system is increased by the respiration of the functional strains, the properties of the phenolic compounds are not very stable, and the phenolic compounds are very easy to decompose when the environmental temperature is increased. Furthermore, the antioxidant activity Of buckwheat sour soup is also closely related to polyphenol flavonoids, And the TingtingSong et al (published by Song et al in 2020 Journal Of Food Science And Technology-Mysore 31(2): 44-53) study showed that grain fermentation can produce more bioactive compounds And antioxidants, Bajalan et al (published by Bajalan et al in 2016 in Industrial Crops And Products 87(3): 255-260) demonstrated that different phenolic contents are the main factors determining the antioxidant capacity Of flavone extracts, And found that there is a linear relationship between total phenolic aldehyde compounds And antioxidant activity (published by Corral-Aguayo et al in 2008 Of Journal Of Agricultural chemicals And Chemistry 56(22): 10498; published by Cornal-guana 1153 in Found 1163). Research on Zhao Xiao Juan and the like (refer to Zhao Xiao Juan and the like, published in 2014 in food science 35(13):122- & 126) finds that the total flavonoids and the total phenols in the tartary buckwheat natto sauce have obvious correlation with the antioxidant activity.

Table 4: differential metabolite table of main polyphenols before and after buckwheat fermentation

Data processing

All mass spectrometry data acquisition and target compound quantitative analysis Work were done by SCIEX analysis Work Station Software (version 1.6.3). The mass spectrum was originally converted to TXT format using MSconventer software. And then the R program package is combined with a self-built database to complete work such as peak extraction, annotation and the like. Data were formatted Centrally (CTR) using SIMCA-P14.1 software and then subjected to automated modeling analysis. The construction of the metabolic pathway was performed by MetabioAnalyst 4.0(https:// www.metaboanalyst.ca). Excel2020 software test data was used for mapping, and statistical correlation of multiple comparisons (Duncan's new polarization difference test) was carried out using SPSS17.0 (note: BCN in all figures represents blank experiment, buckwheat rice flour culture medium without inoculation; BY represents inoculation of Kluyveromyces marxianus YK2021-2, BL represents inoculation of Lactobacillus casei LB20201, BY: L represents mixed fermentation of the two, and the result is assigned with the

numbers

12, 36 and 60 for fermentation time h).

Table 5: main experimental reagent

Table 6: main experimental instrument

Instrument for measuring the position of a moving object	Company(s)
		MJ-160B-II constant temperature incubator	Shanghai leap-in medical instrument factory
BXM-30R vertical pressure steam sterilization pot	Shanghai Bowen realty Co Ltd medical equipment factory
		XL-06A small size pulverizer	Shanghai Zhikai powder machinery manufacturing Co., Ltd
ZQPL-200 constant temperature shaking table	Leiboteri instruments and Equipment Co, Tianjin
		PHS-3CpH meter	Shanghai rainbow instruments & meters Co., Ltd
Multifunctional microplate reader of petri Maxid3	Milli molecular instruments (Shanghai) Co., Ltd
		ECLIPSE-E200 microscope	Nikon corporation
1260 high performance liquid chromatograph	Agilent Technologies Co., Ltd.
		ExionLCAD ultra-high performance liquid phase	Sciex Corp
QTrap6500+ high sensitivity mass spectrum	Sciex Corp
		Heraeus fresco17 centrifuge	THERMO FISHER SCIENTIFIC Inc.
TQ8040 gas chromatography-mass spectrometer	SHIMADZU ENTERPRISE MANAGEMENT (CHINA) Co.,Ltd.

In conclusion, the functional microorganisms are applied to the buckwheat, so that the buckwheat sour soup is sour and refreshing in taste and rich in flavor, the contents of organic acids and volatile flavor compounds are obviously increased, and the mixed fermentation result is superior to that of single-strain fermentation; the buckwheat is fermented by the functional microorganisms, so that the antioxidant activity of the buckwheat is increased, the content of functional components is improved, and a certain theoretical basis is provided for the application of the functional microorganisms and the development of buckwheat fermented food.

The foregoing is illustrative of the preferred embodiments of this invention, and it is to be understood that the invention is not limited to the precise form disclosed herein and that various other combinations, modifications, and environments may be resorted to, falling within the scope of the concept as disclosed herein, either as described above or as apparent to those skilled in the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Guizhou university

<120> a functional microorganism, and preparation method and application thereof

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<213> Kluyveromyces marxianus (YK 2021-2) (Kluyveromyces marxianus)

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gcgtgctcga gtatgatgat agattgctgg gggatcgtct gaacaaggcc tgcgcttaat 60

tgcgcggcca gttcttgatt ctctgctatc agttttctat ttctcatcct aaacacaatg 120

gagttttttc tctatgaact acttccctgg agagctcgtc tctccagtgg acataaacac 180

aaacaatatt ttgtattatg aaaaactatt atactataaa atttaatatt caaaactttc 240

aacaacggat ctcttggttc tcgcatcgat gaagaacgca gcgaattgcg atatgtattg 300

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gtggtaagct tgggtcaaag agactcatag gtgttataaa gactcgctgg tgtttgtctc 600

cttgaggcat acggctttaa ccaaaactct caaagtttga cctcaaatca ggtaggagta 660

cccgctgaac ttaagcatat caataagccg gaggaa 696

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<213> Lactobacillus casei (LB 20201) (Lactobacillus casei)

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aaccatgatc taggctgttt ttttcatctt gtcacttaac ggctcctcct aaagggttac 60

gccaccggct tcgggtgtta caaactctca tggtgtgacg ggcggtgtgt acaaggcccg 120

ggaacgtatt caccgcggcg tgctgatccg cgattactag cgattccgac ttcgtgtagg 180

cgagttgcag cctacagtcc gaactgagaa tggctttaag agattagctt gacctcgcgg 240

tctcgcaact cgttgtacca tccattgtag cacgtgtgta gcccaggtca taaggggcat 300

gatgatttga cgtcatcccc accttcctcc ggtttgtcac cggcagtctt actagagtgc 360

ccaactaaat gctggcaact agtcataagg gttgcgctcg ttgcgggact taacccaaca 420

tctcacgaca cgagctgacg acaaccatgc accacctgtc attttgcccc cgaaggggaa 480

acctgatctc tcaggtgatc aaaagatgtc aagacctggt aaggttcttc gcgttgcttc 540

gaattaaacc acatgctcca ccgcttgtgc gggcccccgt caattccttt gagtttcaac 600

cttgcggtcg tactccccag gcggaatgct taatgcgtta gctgcggcac tgaagggcgg 660

aaaccctcca acacctagca ttcatcgttt acggcatgga ctaccagggt atctaatcct 720

gttcgctacc catgctttcg agcctcagcg tcagttacag accagacagc cgccttcgcc 780

actggtggtc ttccatatat ctacgcattt caccgctaca catggagttc cactgtcctc 840

ttctgcactc aagtttccca gtttccgatg cgcttcctcg gttaagccga gggctttccc 900

atcagactta aaaaaccgcc tgcgctcgct ttacgcccaa taaatccgga taacgcttgc 960

cacctacgta ttaccgcggc tgctggcacg aagtaacccg ggctttctgg ttggataccg 1020

tcacgccgac aaa 1033

Claims

1. A functional microorganism characterized by: comprises Kluyveromyces marxianus YK2021-2 and Lactobacillus casei LB 20201; the Kluyveromyces marxianus YK2021-2(Kluyveromyces marxianus) is deposited in the Guangdong province microorganism strain collection center at 7-5 months in 2021, and the deposit number is GDMCC NO: 61737, respectively; the Lactobacillus casei LB20201(Lactobacillus casei) has been deposited at Guangdong provincial microorganism culture Collection No. GDMCC No. 61034 at 6-14.2020.

2. A functional microorganism according to claim 1, wherein: the volume ratio of the Kluyveromyces marxianus YK2021-2 to the Lactobacillus casei LB20201 is 1: 5.

3. A functional microorganism according to claim 1, wherein: the screening method of the Kluyveromyces marxianus YK2021-2 comprises the following steps:

then extracting a sample from a single bacterial colony for sequencing, carrying out homology comparison, and simultaneously constructing a bacterial strain phylogenetic tree;

4. A method for preparing a functional microorganism, which is characterized by comprising the following steps: the method comprises the following steps:

mixing Kluyveromyces marxianus YK2021-2 and Lactobacillus casei LB20201 at a volume ratio of 1:5 to form a functional microorganism, wherein the Kluyveromyces marxianus YK2021-2 and the Lactobacillus casei LB20201 are activated twice.

5. The method for producing a functional microorganism according to claim 4, wherein: the initial bacterial liquid concentrations of the Kluyveromyces marxianus YK2021-2 and the Lactobacillus casei LB20201 are both 1 × 10 ⁸ CFU/mL。

6. Use of a functional microorganism, characterized in that: the functional microorganism is used for improving the functional components of the buckwheat sour soup in the fermentation process.

7. The method according to claim 6, wherein the step of preparing the functional microorganism comprises: the preparation method of the buckwheat sour soup comprises the following steps:

s1: sample preparation

S11, preparing buckwheat flour: weighing dried buckwheat seeds, crushing, and sieving by a 80-mesh sieve for later use;

s2: fermentation of

Inoculating the functional microorganism of claim 4 in the buckwheat flour liquid medium obtained in step S12, wherein the volume of the functional microorganism is 4% of the volume of the buckwheat flour liquid medium, and fermenting at 33 deg.C for 74 h.